Pair of Chinese cabbage dominant complementary clubroot-resistant gene localization and close linkage SSR molecular markers and application thereof

Abstract

The invention discloses a pair of Chinese cabbage dominant complementary clubroot-resistant gene localization and close linkage SSR molecular markers and application thereof, and belongs to the field of crop breeding and biomolecular genetics. According to the method, a disease-resistant material 'CR-20' and a susceptible material 'BJN3-1' are selected to be hybridized, disease-resistant genes contained in the disease-resistant material are preliminarily positioned by constructing an F2-generation genetic segregation population, and disease-resistant sites are finally positioned between an A01: SZC-7 marker and an LSN-3-115 marker and between an A08: ACMP00373 marker and an SZC-37 marker by adopting a genetic map positioning method and combining a BSA sequencing method. The two disease-resistant sites show a dominant complementary genetic relationship. According to the invention, the molecular marker closely linked with the anti-disease gene contained in the 'CR-20' is found, the initial positioning of the site is completed, technical support is provided for carrying out anti-clubroot transformation work of brassica crops by utilizing the 'CR-20' material in the future, and more choices are provided for anti-clubroot breeding research of Chinese cabbage and other brassica crops in China.

Description

Technical Field

[0001] The invention relates to the field of crop breeding and biomolecular genetics, and in particular to the positioning of a pair of dominant complementary clubroot resistance genes in cabbage and closely linked SSR molecular markers and applications thereof. Background Art

[0002] Clubroot, also known as the "cancer of cruciferous plants", is a highly contagious soil-borne disease caused by Plasmodiophora brassicae. Plasmodiophora brassicae specifically parasitizes the roots of living plants in the Brassica family, such as Brassica. When the root system is infected by Plasmodiophora, the thin-walled cells divide and increase, resulting in the formation of a large number of tumors in the root system of the plant, which hinders the physiological function of the root system, causing the plant to grow slowly, wilt or die. In recent years, the main production areas of Brassica vegetables and oil crops such as cabbage, kale, and rapeseed have been severely damaged by clubroot, and there is a trend of large-scale spread, which seriously restricts the planting and production of Brassica crops.

[0003] Chinese cabbage (Brassica rapa ssp.pekinensis, A genome, 2n=20) is an important leafy vegetable crop of the genus Brassica. However, cabbage clubroot is currently a serious threat. Currently, the main methods for preventing and controlling clubroot are chemical, physical, biological, and disease-resistant breeding. Planting disease-resistant varieties is a safe, green and effective measure to control the disease. Therefore, screening and identifying disease-resistant germplasm resources, studying the genetic basis of clubroot resistance, exploring excellent disease-resistant alleles and analyzing their disease resistance mechanisms are of great significance to promoting the breeding of cabbage varieties resistant to clubroot.

[0004] Clubroot resistance (CR) genes are mostly derived from Brassica rapa (Piao et al., 2007; 2009). The most widely used clubroot resistant materials are the ECD series of Brassica rapa, especially ECD01, ECD02, ECD03 and ECD04 (Hasan et al., 2021). The CR loci of Brassica rapa are mainly distributed in the A genome. So far, the CR loci identified from Brassica rapa include Crr1 (A08), Crr2 (A01), Crr3 (A03), Crr4 (A06), CRa (A03), CRb (A03), CRc (A02), CRk (A02), Rcr1 (A03), PbBa3.1 (A03), PbBa3.2 (A03), PbBa3.3 (A03) and PbBa8.1 (A08). Among them, the CRb gene is widely used in the breeding of disease-resistant cabbage varieties in my country. However, the clubroot CR gene has physiological race specificity. Due to physiological race changes and the effect of selection pressure, disease-resistant varieties containing a single CR gene are prone to loss of resistance during production. Therefore, the continuous discovery of new disease-resistant genes is the key to the continuous, safe and effective prevention and control of clubroot. By constructing recombinant inbred lines to locate disease-resistant genes and developing molecular markers linked to disease-resistant genes, it has great application value for breeding disease-resistant varieties and shortening the breeding period. Summary of the invention

[0005] The purpose of the present invention is to provide a pair of cabbage dominant complementary clubroot resistance gene positioning and tightly linked SSR molecular markers and their application, so as to solve the problems existing in the above-mentioned prior art. The disease-resistant genes contained in the resistant material 'CR-20' are genetically analyzed and preliminarily positioned by using the genetic map positioning method combined with the BSA sequencing method, and finally the disease-resistant locus is located between the A01: SZC-7 marker and the LSN-3-115 marker, named BraPb1.3; A08: between the ACMP00373 marker and the SZC-37 marker, named BraPb8.4. There is a dominant complementary relationship between the two disease-resistant loci contained in 'CR-20'.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an SSR molecular marker tightly linked to a cabbage clubroot resistance gene, wherein the disease resistance locus of the SSR molecular marker is located between the SZC-7 marker and the LSN-3-115 marker on the cabbage chromosome 1 (A01), and between the ACMP00373 marker and the SZC-37 marker on the chromosome 8 (A08);

[0008] The primer pair used to amplify the SZC-7 marker is shown in SEQ ID NO: 1-2;

[0009] The primer pair used to amplify the LSN-3-115 marker is shown in SEQ ID NO: 3-4;

[0010] The primer pair used to amplify the ACMP00373 marker is shown in SEQ ID NO: 5-6;

[0011] The primer pair used to amplify the SZC-37 marker is shown in SEQ ID NO: 7-8.

[0012] The invention also provides a kit for identifying the cabbage clubroot resistance gene, comprising a primer pair for amplifying the SZC-7 marker, the LSN-3-115 marker, the ACMP00373 marker and the SZC-37 marker.

[0013] The present invention also provides application of the SSR molecular marker or the kit in identifying cabbage clubroot resistance genes.

[0014] The present invention also provides application of the SSR molecular marker or the kit in screening cabbage clubroot resistant materials.

[0015] The present invention also provides application of the SSR molecular marker or the kit in molecular marker-assisted breeding of Chinese cabbage.

[0016] The present invention also provides a method for identifying a cabbage clubroot resistance gene, comprising the following steps:

[0017] The genomic DNA of the cabbage to be tested is amplified using a primer pair for amplifying the SZC-7 marker, the LSN-3-115 marker, the ACMP00373 marker and the SZC-37 marker, the genotype of the allele amplified by the primer pair is analyzed, and whether the cabbage material to be tested has a clubroot resistance gene is determined according to the genotype result.

[0018] Preferably, if both pairs of alleles amplified by the primer pair are dominant, the Chinese cabbage material to be tested has a clubroot resistance gene.

[0019] Preferably, the amplification reaction system is: 10 μL of amplification premix, 2 μL of DNA template, 1 μL each of upstream primer and downstream primer, and 6 μL of ddH2O.

[0020] Preferably, the reaction procedure of the amplification is: 94°C for 7 min; 94°C for 2 min; 55°C for 30 s, 72°C for 30 s, 72°C for 5 min; and hold at 4°C.

[0021] The present invention discloses the following technical effects:

[0022] The present invention has experimentally found that the parent 'CR-20' exhibits disease resistance to the root knot fungus SXTB104, the parent 'BJN3-1' exhibits disease susceptibility to the root knot fungus SXTB104, and the hybrid offspring F1 exhibits disease resistance after inoculation with SXTB104, therefore, the disease resistance gene contained in the resistant parent material 'CR-20' is dominant. The results after the F2 population was inoculated with SXTB104 showed that the ratio of the number of disease-resistant to disease-susceptible individual plants was 9:7, therefore, the disease resistance trait is controlled by two pairs of alleles, and the material exhibits resistance only when both pairs of genes are dominant, proving that there is a dominant complementary relationship between the two loci.

[0023] The disease resistance genes contained in the resistant material 'CR-20' were analyzed and initially located by combining the genetic map positioning method with the BSA sequencing method. Finally, the disease resistance loci were located between the markers SZC-7 and LSN-3-115 in A01 and named BraPb1.3; and between the markers ACMP00373 and SZC-37 in A08 and named BraPb8.4. 2:3 The association analysis of population phenotype and genotype confirmed that there was a dominant complementary relationship between the two disease resistance loci contained in 'CR-20'. The present invention has completed the preliminary positioning of the disease resistance loci of 'CR-20' and developed molecular markers closely linked to the two loci. The two-loci linkage markers can be directly used for the clubroot resistance gene transfer of 'CR-20'. The present invention provides technical support for the future use of 'CR-20' materials to carry out clubroot resistance gene transfer, and provides more options for the research on clubroot resistance breeding of Chinese cabbage and other Brassica crops in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 A research technology roadmap for the molecular markers of the present invention;

[0026] Figure 2 The results of the identification of the disease resistance of the parents; 1 is the susceptible parent BJN3-1, 2 is the resistant parent CR-20;

[0027] Figure 3 This is the result of F1 disease resistance identification;

[0028] Figure 4The results of CRa disease resistance gene verification; in the left half, 0 represents 5000bp Marker, 1 represents 222 (CRa), 2 represents BJN3-1, and 3 represents CR-20; the right half is the secondary verification, and the order is the same as the left half;

[0029] Figure 5 The results of CRb and CRd disease resistance gene verification; A is the CRb verification result, the linkage markers are TCR01 and TCR05 (Piao et al, 2004), DNA from left to right: 1 represents BJN3-1, 2 represents 222 (CRb), and 3 represents CR-20; B is the CRd verification result, the linkage markers are yau376 and yau106 (Fu Pengyu, 2019), DNA from left to right: 1 represents BJN3-1, 2 represents 85-74 (CRd), and 3 represents CR-20;

[0030] Figure 6 The results of F2 disease resistance identification; from left to right: CK, grade 0, grade 1, grade 2, grade 3;

[0031] Figure 7 It is the BSA sequencing trait association interval positioning map;

[0032] Figure 8 This is the genetic map of chromosome A08;

[0033] Fig. 9 This is the genetic map of chromosomes A01-A03;

[0034] Fig.10 This is the genetic map of chromosomes A04-A06;

[0035] Fig.11 This is the genetic map of chromosomes A07, A09 and A10;

[0036] Fig.12 F 2:3 Results of family disease index analysis;

[0037] Fig.13 The distribution of disease resistance loci obtained by composite interval mapping method; A01 represents chromosome 1, and A08 represents chromosome 8.

[0038] Fig.14 This is the electrophoresis of the amplified products of the tightly linked markers SZC-7 and LSN-3-115 located on the chromosome of A01 between the resistant and susceptible parents; P1 is the susceptible parent BJN3-1, P2 is the resistant parent CR-20, and F2 represents some individual plants of the F2 population;

[0039] Fig.15This is the electrophoresis diagram of the amplified products of the tightly linked markers SZC37 and ACMP00373 located on the chromosome of A08 between the resistant and susceptible parents; P1 is the susceptible parent BJN3-1; P2 is the resistant parent CR-20; F2 represents some individual plants of the F2 population. DETAILED DESCRIPTION

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0043] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0044] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0045] Example 1

[0046] 1. Test materials

[0047] The selected test materials are all from the Vegetable Molecular Laboratory of the College of Horticulture, Shenyang Agricultural University. The disease-resistant material 'CR-20' was selected as the male parent, and the susceptible material 'BJN3-1' was selected as the female parent.

[0048] Test strains: The root-knot bacteria 'SXTB104' was selected as the test strain. The bacterium was identified by the SCD identification method (Pang et al., 2020), and the results showed that the bacterium belonged to the Pb8 physiological race (currently stored in the strain storage refrigerator of the Vegetable Molecular Laboratory of Shenyang Agricultural University).

[0049] 2. Test methods

[0050] The hybridization of the disease-resistant material 'CR-20' and the susceptible material 'BJN3-1' and multi-generation self-pollination were used to construct F 2 groups and F 2:3 The disease resistance gene contained in Chinese cabbage 'CR-20' was preliminarily located by using BSA sequencing combined with traditional genetic map positioning method. Figure 1 .

[0051] 2.1 Method of inoculation of root knot fungus

[0052] The Pb8 physiological race 'SXTB104' was selected as the test strain for later inoculation and identification. This strain will not cause disease in 'CR-20', but 'BJN3-1' showed susceptibility to the disease when inoculated with this strain. The inoculation method is the bacterial liquid injection method. The results of many years of experiments in this laboratory show that this method is not only simple and convenient to operate, but also has relatively accurate inoculation and identification results. The specific inoculation experimental method is as follows:

[0053] 2.1.1 Preparation of bacterial solution

[0054] 1) Take out 50 g of SXTB104 bacterial blocks from the special storage refrigerator for bacterial strains and thaw them at room temperature. After the bacterial blocks are thawed, put them into a wall breaking machine and add 200 mL of sterile water to fully grind them. Filter the mixed liquid with 8 layers of medical sterile gauze, and pour the filtered bacterial liquid into a sterilized volumetric flask.

[0055] 2) Use a pipette to draw the bacterial solution into a 50 mL centrifuge tube, place it in a centrifuge and balance it. Set the program to centrifuge at 4°C and 1000g for 10 minutes. Discard the supernatant and add sterile water. Repeat 3 times.

[0056] 3) Mix the treated bacterial solution thoroughly on a vortex shaker, pipette 100 μL of the bacterial solution into a 1.5 mL centrifuge tube, then add 900 μL of sterile water and shake up and down for 30 seconds. Pipette 100 μL of the diluted bacterial solution into a 1.5 mL centrifuge tube, add 200 μL of aniline blue solution and 700 μL of sterile water, mix well, and stain for 5 minutes. After staining, use a hemocytometer to measure the concentration of the bacterial solution and determine the concentration of the bacterial solution at 1×10 7 pc·mL -1 , and then store in a 4°C refrigerator. When conducting inoculation tests, try to grind the samples on the same day they are used to avoid long-term storage.

[0057] 2.1.2 Inoculation of root knot fungi

[0058] (1) In March 2021, the parents, F1 generation, and 229 F2 generation and F 2:3 The family was sown in a sterilized 72-hole tray and placed in the No. 3 greenhouse of Shenyang Agricultural University (20℃-25℃) for watering and pest control. After 20 days, when the plants had two leaves and one heart, they were inoculated with bacterial liquid injection.

[0059] (2) Prepare a sterilized 5 mL syringe and beaker, and inoculate each plant with 2 mL of bacterial solution. Water the plants the next day after inoculation, with a photoperiod of 16 h light / 8 h dark. Observe the growth of the plants every day to ensure that the soil remains moist.

[0060] (3) Phenotypic identification: Disease resistance identification is carried out 45 days after inoculation. The identification time is adjusted appropriately according to the disease situation of the susceptible parent material. When conducting the inoculation result identification, the soil moisture can be appropriately reduced to ensure the accuracy of the disease results. Use clean water to wash away the excess matrix at the roots of the plants. During the washing process, try to ensure that the roots are intact and clearly visible. Observe the disease situation and conduct graded statistics.

[0061] 2.2 Identification of disease grade

[0062] In May 2021, seven weeks after inoculation, the disease resistance of the test materials was investigated. The disease site caused by root knot fungi is at the root of the plant. Therefore, when identifying the disease, the plants in the plug tray should be gently pulled out and gently washed with water. The treated plants were graded and counted according to the grading method proposed by Buczacki et al. in 1975. The specific standards of this grading method are shown in Table 1 below:

[0063] Table 1 Classification standard of clubroot

[0064]

[0065] 2.3 Group Construction

[0066] The disease-resistant material 'CR-20' was hybridized with the susceptible material 'BJN3-1' to obtain the F1 population. The harvested F1 population was sown in 72-hole trays. After natural vernalization, 5 robust plants were randomly selected and planted in the No. 3 shed experimental base of the College of Horticulture of Shenyang Agricultural University. 200 seeds of the F2 population harvested by artificial pollination were randomly selected and placed in a sterilized culture dish. Sterile water was added to the culture dish until the filter paper in the culture dish was completely soaked. The seeds were protected from light and transferred to a laboratory special refrigerator for vernalization after small buds grew. The temperature was controlled at 4°C (regular watering, light / darkness of 18h / 6h).

[0067] The vernalized seedlings were sown in 72-hole trays, and when they grew to a suitable height, they were planted in the No. 3 shed experimental base of the College of Horticulture of Shenyang Agricultural University. F 2:3 Family lineage.

[0068] 2.4 DNA extraction and concentration detection

[0069] The method for extracting leaf DNA in this experiment is a modified version of the CTAB method. The DNA extracted by this method has the advantages of high concentration and long storage time. The specific experimental steps are as follows:

[0070] I. Pre-cool anhydrous ethanol and 70% ethanol in a -20℃ refrigerator. Take fresh leaves with a length and width of about 1 cm and put them in a 2mL centrifuge tube. Add sterilized steel balls. Arrange the plant number and the centrifuge tube number on the operation board one by one. Wear anti-freeze gloves and transfer the sample to a foam box filled with liquid nitrogen for freezing. The freezing time is 5 minutes. Then take out the operation board and put it horizontally into the vibration grinder. The grinding time is 5 minutes. According to the freezing effect and the size of the sample, perform secondary vibration grinding as appropriate.

[0071] II. Place the ground sample on the laboratory bench and wait for it to cool to room temperature. Use a pipette to add 600 μL of 2% CTAB extraction buffer that has been preheated in a 65°C water bath for 40 minutes. To ensure the uniformity of the mixed solution, shake the operating plate upside down after adding the CTAB extraction buffer.

[0072] III. Fix the mixed samples on the floats in the order of the labels and place them in a 65℃ water bath for 45 minutes. During this period, take out the floats and shake them every 10 minutes to ensure that the samples are heated evenly.

[0073] IV. Prepare chloroform and isoamyl alcohol, wear a gas mask and prepare chloroform in a fume hood at a volume ratio of 24:1. After the water bath is over, insert the samples into the operation plate according to the serial number, and avoid cross contamination during the opening process. Add 600μL of chloroform, shake up and down for 20s, and then transfer to a centrifuge for centrifugation for 15min at 12000r / min.

[0074] V. After centrifugation, use a 1000μL pipette to draw 450μL of supernatant into a 1.5mL centrifuge tube. During the drawing process, the centrifuge tubes of different specifications correspond to each other according to the numbers, and the pipette tips are replaced in time to avoid the inhalation of impurities. After the supernatant is drawn, add 900μL of pre-cooled anhydrous ethanol. After checking that there is no leakage or omission, place the sample horizontally in a -20℃ refrigerator and let it stand for more than 40 minutes. After standing, transfer it to a centrifuge with a program of 12000r / min and centrifuge for 10 minutes.

[0075] VI. After centrifugation, take out the sample, discard the supernatant in the centrifuge tube, and then add 600 μL of pre-cooled 70% ethanol with a 1000 μL pipette. After the drug is added, centrifuge it in a centrifuge at a program of 12000 r / min for 5 minutes.

[0076] VII. After centrifugation, discard the supernatant in the centrifuge tube. Place the centrifuge tube containing DNA horizontally in a fume hood to air dry for 24 hours. After ventilation, add 200 μL of ultrapure water and dissolve at room temperature for 2 hours. Then use an enzyme reader or ultraviolet spectrophotometer to detect the DNA concentration.

[0077] 2.5 Polymorphic marker screening

[0078] After verifying that the extracted DNA meets the criteria for further testing, polyacrylamide gel electrophoresis combined with SSR markers and Indel markers developed based on the cabbage genome (Ramchiary et al., 2011; Jiao Yang, 2022) was used to screen primers that meet the polymorphism between the parents. The screened primers should cover 10 chromosomes and the primers on each chromosome should be evenly distributed, so as to use polymorphic primers to identify the genotype of F2 generation single plants and construct a genetic map. The synthesis of all primers was completed by Suzhou Hongxun Company. The entire experimental steps for polymorphic marker screening are as follows:

[0079] The PCR amplification in the present invention adopts a 20uL system, and the addition amount of each drug and the reaction procedure are shown in Table 2 below:

[0080] Table 2 PCR amplification system

[0081]

[0082] The PCR reaction program is shown in Table 3:

[0083] Table 3 PCR amplification program

[0084]

[0085] The amplified products were detected by polyacrylamide gel electrophoresis.

[0086] 3. BSA sequencing and QTL analysis

[0087] 3.1 BSA sequencing

[0088] According to the results of clubroot disease identification in the F2 population, 40 extremely resistant and 40 extremely susceptible plants were selected from the 229 F2 populations to construct disease-resistant pools (R pool) and susceptible pools (S pool). DNA from the two pools and parents was extracted and BSA sequencing was completed by Shanghai Paisono Biotechnology Co., Ltd. The main steps of genome BSA sequencing are as follows:

[0089] i. Extraction of total genomic DNA;

[0090] ii. Sequencing library preparation;

[0091] iii. Perform high-throughput sequencing on a machine;

[0092] iv.Data quality control;

[0093] v. Align with reference genome;

[0094] vi.SNP, Indel detection and annotation;

[0095] vii. Target trait positioning;

[0096] viii. Target trait region analysis.

[0097] 3.2 Construction of genetic map

[0098] After screening and counting the primers with different banding patterns between the parents, further verify whether there is still polymorphism in the F2 population and record the genotype. The genotype consistent with the susceptible parent 'BJN3-1' is recorded as 'a', the genotype consistent with the resistant parent 'CR-20' is recorded as 'b', and the heterozygous band is recorded as 'h'. The software joinmap 4.0 was used to construct the genetic map, the LOD value was set to 2.0, and kosambi's was selected as the mapping function to verify whether the selected markers are linked. The constructed genetic map should cover 10 chromosomes and be evenly distributed on each chromosome.

[0099] 3.3 QTL analysis

[0100] After the map is constructed, combined with F 2:3 The disease index was calculated based on the identified disease conditions using the formula: disease index = ∑ (number of diseased plants at each level × corresponding level) / total number of plants in the survey population × highest level value. Disease indexes ≥ 25 were recorded as susceptible, and disease indexes less than 25 were recorded as resistant. The F2 phenotype was inferred based on the calculation results, and QTL analysis was performed using the software QTLIciMapping.

[0101] 4. Results and Analysis

[0102] 4.1 Genetic analysis

[0103] 4.1.1 Identification of disease resistance of parents and F1

[0104] The two parents, 'CR-20' and 'BJN3-1', and F1 were inoculated and identified. The results are as follows Figure 2-Figure 3As shown, the results showed that 36 'CR-20' trees were resistant to the disease, 36 'BJN3-1' trees were susceptible to the disease, and 30 F1 trees were resistant to the disease. This result shows that the disease resistance trait of 'CR-20' is controlled by a dominant gene.

[0105] In order to further verify whether 'CR-20' contains known disease-resistant genes, the DNA fragments of materials containing the disease-resistant genes and the DNA fragments of 'BJN3-1' were amplified using the CRa, CRb, and CRd linkage markers available in the laboratory. The disease-resistant material '222' was used to verify CRa and CRb, and the disease-resistant material '85-74' was used to verify CRd. CRa was verified by agarose gel electrophoresis, and CRb and CRd were verified by polyacrylamide gel electrophoresis. Figure 4-Figure 5 As shown, the results showed that 'CR-20' did not contain the above-mentioned known disease resistance gene fragments.

[0106] 4.1.2 Identification of disease resistance in F2 population

[0107] The results of inoculation of 229 F2 plants showed that the ratio of disease resistance to disease sensitivity was 138:91, which was compared with the theoretical value of 9:7 by chi-square test (see Table 4). 2 =1.28 <x 2 (0.05), which is not significantly different from the theoretical value and is consistent with the 9:7 separation ratio. Figure 6 The results showed that the resistance of 'CR-20' to root knot fungus was controlled by two genes and the resistance was exhibited only when both genes were dominant.

[0108] Table 4F2 Chi-square test

[0109]

[0110] 4.2 BSA sequencing data analysis

[0111] 4.2.1 GPS Identification of Genomic Regions

[0112] GradePool-Seq is a new quantitative trait gene localization method developed by Han Bin's research group. The study proposed a quantitative trait locus (QTL) mapping method, which is GradedPool-Seq (GPS) for rapid mapping of QTLs. It is based on genome sequencing analysis of the gradient pool samples of the F2 offspring, followed by BSA on the machine. Theoretically, the lower the P value, the higher the correlation between the variant and the phenotype. However, due to the presence of a large amount of background noise in the SNP / Indel signal due to distant genetic hybridization, the reduction of background noise must be considered. The noise reduction algorithm implemented in the study is a non-overlapping sliding window method, that is, within a certain genomic interval (about 400kb), the ratio of the number of variants that significantly exceed the threshold to the total number of variants is calculated. The interval with the largest ratio, that is, the SNPs / Indels group with significant p-values, has the highest proportion. After adopting the strategy of reducing background noise, the results are presented in the form of a ratio graph. The place with a high ratio is the trait association interval, see Figure 7 The candidate interval was located at 9.8Mb-12.2Mb on chromosome A08 and was named BraPb8.4 (Table 5).

[0113] Table 5 BSA candidate intervals

[0114]

[0115] 4.3 Construction of F2 genetic map

[0116] After genetic analysis of 'CR-20' and 'BJN3-1' and their constructed populations, the present invention concluded that the disease resistance trait was controlled by two dominant complementary genes, while BSA only detected a signal at one locus. Therefore, the present invention constructed a genetic map of the material and used the QTL positioning method to determine the disease resistance loci located on other chromosomes.

[0117] 4.3.1 Screening of polymorphic markers between parents

[0118] SSR markers and Indel markers developed based on the cabbage genome were used to screen markers with polymorphism between parents, and markers with clear bands, obvious polymorphism and co-dominance were selected for analysis of the F2 population and subsequent experiments.

[0119] 4.3.2 F2 population marker verification and map construction

[0120] The BSA test results identified a disease-resistant locus located at 9.8Mb-12.2Mb on chromosome A08. Therefore, the markers on chromosome A08 were first analyzed, and the screened markers on chromosome A08 were used to verify the F2 population. There were 13 pairs of SSR markers and 2 pairs of Indel markers used to construct a genetic map. The software Joinmap 4.0 was used for analysis, and the LOD threshold was set to ≥2.5. The kosambi algorithm was used, and the analysis results showed that 5 pairs of markers failed to link. Subsequently, SSR primers were designed using the software SSR hunter according to the candidate interval given by BSA, and the number of linked markers in the candidate interval was increased. A pair of primers was designed every 30Wbp, and a total of 30 pairs of primers were designed. This part of the markers was screened, and the markers that met the requirements were added to the genetic map. The final genetic map is as follows. Figure 8 As shown, the genetic map is 42.3 cM in length, with 16 pairs of markers, including one pair of Indel markers, an average map distance of 2.64 cM, and a density of 0.38 / cM.

[0121] After completing the genetic map on chromosome A08, continue to screen primers with polymorphisms between parents on the remaining chromosomes, and try to ensure that there are 10 linked markers in each map. After the genetic maps on the remaining chromosomes are constructed using the same method, they are named in the order of A01 to A10. The genetic maps on the remaining chromosomes are as follows: Figure 9-11 As shown:

[0122] All the constructed genetic maps were analyzed as a whole, and the results are shown in Table 6. The results showed that the map was 564.5cM in length, with an average map distance of 5.82cM and a density of 0.17cM. Among them, the molecular marker density on chromosome A08 was the largest, at 0.38 / cM, and the molecular marker density on chromosome A10 was the smallest, at 0.08 / cM; from the average map distance, the distance on chromosome A10 was the largest, at 12.7cM, and the distance on chromosome A08 was the smallest, at 2.64cM; in general, the markers on chromosome A10 were the most uniform, and the genetic map distance of chromosome A03 was the longest, at 73.2cM.

[0123] Table 6 Genetic linkage map analysis

[0124]

[0125]

[0126] 4.4 QTL analysis results

[0127] 4.4.1F 2:3 Calculation of disease index

[0128] Statistics good F 2:3After the disease situation of the family was analyzed, the formula was used: ∑ (number of diseased plants at each level × corresponding level) / total number of plants in the survey group × the highest level value; F 2:3 The family disease index was analyzed and the results were shown in Table 7. The results showed that: F 2：3 The ratio of disease resistance to disease sensitivity in the population is 116:76, and the chi-square test result is x 2 =1.19 <x 2 (0.05), which was not significantly different from the theoretical value of 9:7 and still met the 9∶7 segregation ratio. This result further confirmed that the disease resistance of 'CR-20' was controlled by two pairs of dominant complementary genes.

[0129] Table 7F 2:3 Chi-square test results

[0130]

[0131] Enter the calculated disease index into SPSS software to analyze the peak value. The results are as follows Fig.12 As shown, the analysis results show that F 2:3 The disease index of the family was skewed normal distribution, which met the basic characteristics of quantitative traits and the basic requirements for further QTL experiments.

[0132] 4.4.2 QTL analysis

[0133] After calculating F 2:3 The family disease index was combined with the constructed genetic map, and the QTL results were analyzed using the software QTL IciMapping, such as Fig.13 As shown in Table 8, the two disease resistance loci BraPb1.3 and BraPb8.4 were finally locked in the 6.3-7.4Mb range of chromosome A01, between markers SZC-7 and LSN-3-115; the 9.9-10.5Mb range of chromosome A08, between markers ACMP00373 and SZC-37, with LOD values ​​of 2.54 and 11.44, respectively, and genetic contribution rates of 4.42% and 23.95%, respectively. (The reference genome of Chinese cabbage is Brara Chiifu V 1.5genome, see www.brassicadb.cn) The electrophoresis diagrams of the four linked markers in the resistant and susceptible materials and F2 plants are shown in Fig.14 and Fig.15 The genotype with the same band pattern as the resistant parent 'CR-20' is the resistant genotype, the genotype with the same band pattern as the susceptible parent 'BJN3-1' is the susceptible genotype, and the genotype with both bands is the heterozygous genotype.

[0134] Table 8 QTL analysis of disease resistance loci in CR-20

[0135]

[0136] 4.4.3F 2:3 Population phenotype and genotype association analysis validation

[0137] To further verify that the disease resistance trait is controlled by two loci, the F 2:3 The phenotype of the family was associated with the F2 genotype, and the genotypes of the four linked markers were counted. The genotypes consistent with the susceptible parent were recorded as A, and the genotypes consistent with the resistant parent were recorded as B. Families with the genotype of the susceptible parent were classified into one category, recorded as Category A, and families without the genotype of the susceptible parent were classified into one category, recorded as Category B. The average disease index of the two parts was calculated respectively. The calculation results are shown in Tables 9 and 10:

[0138] Table 9 Analysis of average disease index of category B

[0139]

[0140]

[0141] Table 10 Analysis of the average disease index of Class A

[0142]

[0143] Note: “-” represents missing genotype.

[0144] The calculation results were analyzed. The average disease index of class A was 40.76, which was higher than 25 and belonged to the highly susceptible type. The average disease index of class B was 9.57, which belonged to the highly resistant type. The ratio of the number of resistant to susceptible families in the two parts was 111:81. The chi-square test result was x 2 =0.13 <x 2 (0.05), which is not significantly different from the theoretical value. This result further proves that the disease resistance of 'CR-20' is controlled by two genes, and the material shows resistance only when both alleles are dominant, further illustrating the dominant interaction relationship between the two loci. In addition, the linkage marker developed based on the two loci in the present invention can be directly used for the breeding of clubroot resistance loci of the disease-resistant material 'CR-20'.

[0145] In summary, the present invention has completed the genetic analysis and preliminary positioning of the 'CR-20' disease resistance locus, and developed molecular markers tightly linked to the two loci. The two-locus linkage markers can be directly used for the transfer of clubroot resistance genes in other Brassica crops.

[0146] Specific information of the literature involved in the above embodiments:

[0147] (1)Pang W, Liang Y, Zhan Z, Li X, Piao Z. Development of a siniticclubroot differential set for the pathotype classification of Plasmodiophorabrassicae. Front Plant Sci, 2020, 11, 568771.

[0148] (2)Piao ZY, Deng YQ, Choi SR, Park YJ, Lim YP. SCAR and CAPS mapping ofCRb, a gene conferring resistance to Plasmodiophora brassicae in Chinesecabbage (Brassica rapa ssp.pekinensis). TheorAppl Genet, 2004, 108, 1458-1465.

[0149] (3) Fu Pengyu, Positional cloning and functional identification of the clubroot resistance gene CRd in Chinese cabbage

D

[0150] (4) Jiao Yang, Localization of clubroot resistance genes based on Chinese cabbage “CR-096” [D]. Shenyang Agricultural University, 2023, 2.

[0151] (5)Ramchiary N, Nguyen VD, Li X, Hong C, Dhandapani V, Choi SR, Yu G, PiaoZY, Lim YP. Relatives.DNAResearch,2011,18,305-320.

[0152] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An SSR molecular marker tightly linked to a cabbage clubroot resistance gene, characterized in that: The disease resistance loci of the SSR molecular marker are located between the SZC-7 marker and the LSN-3-115 marker on chromosome 1 of Chinese cabbage, and between the ACMP00373 marker and the SZC-37 marker on chromosome 8; The primer pair used to amplify the SZC-7 marker is shown in SEQ ID NO: 1-2; The primer pair used to amplify the LSN-3-115 marker is shown in SEQ ID NO: 3-4; The primer pair used to amplify the ACMP00373 marker is shown in SEQ ID NO: 5-6; The primer pair used to amplify the SZC-37 marker is shown in SEQ ID NO: 7-8.

2. A kit for identifying Chinese cabbage clubroot resistance genes, characterized in that: The method comprises a primer pair for amplifying the SZC-7 marker, the LSN-3-115 marker, the ACMP00373 marker and the SZC-37 marker in claim 1.

3. Use of the SSR molecular marker according to claim 1 or the kit according to claim 2 in identifying the clubroot resistance gene of Chinese cabbage.

4. Use of the SSR molecular marker according to claim 1 or the kit according to claim 2 in screening Chinese cabbage resistant to clubroot.

5. Use of the SSR molecular marker according to claim 1 or the kit according to claim 2 in molecular marker-assisted breeding of Chinese cabbage.

6. A method for identifying cabbage clubroot resistance genes, characterized in that: The following steps are involved: The genomic DNA of the cabbage to be tested is amplified using the primer pair for amplifying the SZC-7 marker, the LSN-3-115 marker, the ACMP00373 marker and the SZC-37 marker in claim 1, the genotype of the allele amplified by the primer pair is analyzed, and whether the cabbage material to be tested has a clubroot resistance gene is determined according to the genotype result.

7. The method according to claim 6, characterized in that If both pairs of alleles amplified by the primer pair are dominant, the Chinese cabbage material to be tested has a clubroot resistance gene.

8. The method according to claim 6, characterized in that The amplification reaction system is: 10 μL of amplification premix, 2 μL of DNA template, 1 μL of upstream primer and downstream primer respectively, and 6 μL of ddH2O.

9. The method according to claim 6, characterized in that The reaction procedure of the amplification is: 94°C for 7 min; 94°C for 2 min; 55°C for 30 s, 72°C for 30 s, 72°C for 5 min; and hold at 4°C.

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